We have purified and sequenced pol ORF-encoded proteins from HIV-1-infected cells and purified virus. The p64 and p5l RT (reverse transcriptase) and p34, the putative integrase that maps near the C-terminus of the pol ORF, were sequenced in this manner. We have also sequenced a plO gag-pol protease responsible for the processing of the gag and gag-pol fusion protein. A sensitive assay for the protease using defined oligopeptides has also been developed. Seven human immunodeficiency virus (HIV) gag polypeptides were identified in the extracellular virus and in infected CD4+ lymphocytes by peptide mapping and limited amino acid sequencing. Two gag precursor proteins of 55,000 (p55) and 41,000 (p4l) daltons were rapidly labeled and readily processed into the major internal gag proteins that were aligned within the gag ORF as NH2-pl6(MA)-p24(CA)-p9(NC)-p7-COOH. Myristoylated pl6(MA) protein was processed from the myristoylated p55 gag precursor protein. Both the mature p24(CA) and pl6(MA) proteins were phosphorylated at Ser residue(s). Two forms of gag p4l species, one from the C-terminal processing of p55 and the other from N-terminal processing of p55 or from de novo synthesis initiating at an internal Met at position 142 of the gag ORF were identified. Recombinant vaccinia viruses expressing the HIV-1 protease, p64 RT, p55, and p4l gag precursor proteins with or without protease have been constructed. Protein expression has been confirmed by immunological procedures. Based on the protein sequence data, the DNA encoding integration protein has been constructed and expressed in the vaccinia vector. A 15 kDa C-terminal cleavage product of p64 RT was found to possess RNase H activity, and the DNA corresponding to this protein has also been expressed in vaccinia. All these constructs are now being used to study the biochemical mechanisms of gag-pol processing.